A new player in the biogenesis of lysosome-related organelles

In multicellular organisms, the endolysosomal system consists of a complex network of interconnected membrane compartments that play a crucial role in various cellular functions, such as metabolic regulation, cellular signaling, and innate immunity. The endolysosomal system must adapt to different cellular environments, allowing cells to customize it based on their specific needs. A notable adaptation of the endolysosomal system is lysosome-related organelles (LROs), specialized compartments with unique contents and functions (1). Cells use specific mechanisms to separate the contents of LROs from materials destined for conventional endolysosomes. In humans, there are many types of LROs that coexist with endolysosomes, such as cytolytic granules of resting cytotoxic T cells, melanosomes, Weibel-Palade bodies, and platelet dense and α granules. Disruptions in LRO formation or function can lead to severe disorders like Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, cholestasis syndrome, and gray platelet syndrome (2). With the remarkable progress in recognizing the physiological importance of LROs (2, 3, 4), the identification of a new family of proteins by Li et al. that have a conserved function in LRO formation adds a fresh perspective to the intricate control of this fundamental cellular process.

Gut granules/LROs are unique to worms and serve as storage organelles for zinc and 3-hydroxyanthranilic acid in intestinal cells, contributing to cellular homeostasis and enhancing host immune function (5, 6, Preprint). An unbiased genetic screen in Caenorhabditis elegans identified the LysM domain–containing protein LMD-2 as a crucial regulator of gut granule formation (Fig. 1) (7). GLO-1 is C. elegans’ counterpart to Rab32/38, the Rab GTPases found specifically on LROs in mammals. Only active GTP-bound GLO-1 could alleviate the abnormal enlargement of gut granules seen in lmd-2 mutant worms. However, the overexpression of GLO-1 guanine nucleotide exchange factor (GEF) complex elements GLO-3 and CCZ-1 did not rescue impaired LROs’ biogenesis. This particular phenotype was astutely identified by the researchers, leading to the inference that LMD-2 is crucial for the optimal functioning of the GLO-1–GEF complex and laying the groundwork for subsequent mechanistic investigations.

When delving into the molecular mechanisms, Li et al. found that LMD-2 increases the activity of GLO-3 and CCZ-1, which collectively function as a GEF for GLO-1 (7). The authors utilized a range of techniques to explain the role of LMD-2 in LRO formation. Genetic analysis illustrated that LMD-2 operates upstream of GLO-3, CCZ-1, and GLO-1 (Fig. 1). Biochemical assays uncovered that LMD-2 has a direct interaction with GLO-3, creating a tripartite complex with CCZ-1. This interaction increases the GEF activity of the GLO-3–CCZ-1 complex toward GLO-1 (Fig. 1), leading to its activation. Moreover, the study highlighted the significance of GLO-1 activity in reversing the impairments in LRO formation caused by the absence of LMD-2. The rescue of LRO formation was achieved through the overexpression of wild-type or constitutively active GLO-1, whereas its inactive form did not show the same rescue effect. This observation firmly establishes the functional association between LMD-2 and GLO-1 activation in the development of LROs.

The authors excitingly demonstrate that this regulatory mechanism is conserved in mammals (7). Human homologs of LMD-2, LYSMD1, and LYSMD2 interact with the BLOC-3 complex, the mammalian equivalent of GLO-3/CCZ-1, to enhance its GEF activity toward Rab32. Knocking down both LYSMD1 and LYSMD2 in mouse melanoma cells resulted in enlarged melanosomes and reduced melanin production, similar to the phenotypes seen in C. elegans. To confirm the conservation of this mechanism, the authors conducted cross-species experiments. They found that expressing LYSMD1 or LYSMD2 could rescue LRO defects in C. elegans lmd-2 mutants. Additionally, expressing human Rab32 or its constitutively active form could also alleviate LRO enlargement in lmd-2 animals. These results highlight the evolutionary conservation of the LYSMDs-Rab32 axis in LRO biogenesis and uncover a previously undisclosed regulatory mechanism in melanosome biogenesis. Moreover, this study links observations from a simple model organism to mammalian cells, emphasizing the usefulness of C. elegans in elucidating fundamental cellular processes.

The study by Li et al. complements recent advancements in our understanding of LRO formation (7). For instance, Delevoye et al. highlighted the intricate interactions between different protein complexes, such as BLOC-1, -2, and -3, in melanosome formation (1). This study adds LYSMD proteins to this complex network (Fig. 1), providing more insights into the molecular processes involved in LRO formation. In the broader context of membrane trafficking, this study underscores the complex regulation of Rab GTPases and their GEFs. Recent studies have shown the significance of accessory proteins and posttranslational modifications in modulating GEF activity in various cellular processes (8). The identification of LYSMD proteins as enhancers of GEF activity in LRO formation contributes to this growing body of knowledge and may inspire researchers to explore similar regulatory mechanisms in other vesicle trafficking pathways.

Looking ahead, several interesting aspects emerge from this study. First, it is worth noting that the researchers observed the localization of late-endosomal/lysosomal RAB-7 on enlarged vacuoles in LMD-2–deficient cells, which supports the previously established role of endolysosomes in LRO formation (9). Recently, the Sternberg group conducted a study using single-tissue proteomics to identify proteins specific to gut granules in C. elegans (10). They identified 22 potential candidates, including well-known proteins such as APB-3, PGP-2, and CDF-2. Interestingly, they did not find LMD-2, CCZ-1, and GLO-3, suggesting that the absence of gut granules does not impact the presence of these proteins within cells. Furthermore, Li et al. found that the loss of CCZ-1 or GLO-3 hinders the enlargement of GLO-1–labeled vacuoles in lmd-2 animals. This indicates that these proteins may have additional functions in endolysosomes that contribute to the formation of LROs, a concept that warrants further exploration. Second, delving into the structural aspects of the LYSMD–GEF–Rab32 complex may offer significant insights into how GEF enhancement occurs. Unraveling the molecular intricacies of this interaction has the potential to inform the design of small molecule regulators for LRO biogenesis. Third, by investigating the regulation of LYSMD proteins themselves— whether through transcriptional control, posttranslational modifications, or protein–protein interactions—researchers may uncover additional layers of control in LRO biogenesis. This line of inquiry could illuminate how cells adapt the formation of LROs to different physiological conditions or developmental stages. Finally, the identification of LYSMD proteins as regulators of Rab32 activation suggests that similar mechanisms may also be at play for other Rab GTPases implicated in organelle biogenesis. Exploring this potential could provide a more comprehensive understanding of the cellular mechanisms governing the development and operation of diverse organelles.

In conclusion, the discovery of LYSMD proteins as conserved regulators of LRO formation represents a significant advancement in our understanding of organelle biogenesis. By uncovering a new family of proteins involved in this process, Li et al. not only deepen our knowledge of a fundamental cellular mechanism but also open up possibilities for exciting future research in cell biology and potential therapeutic interventions for LRO-related disorders. As we continue to explore the complexities of organelle formation and function, studies like this serve as a reminder that there are still fundamental discoveries to be made in cell biology, with wide-reaching implications for human health and disease.